EP0776975B1 - Méthode pour l'utilisation du système promoteur de l'ADH-II de levure pour la production biotechnologique de protéins hétérologues - Google Patents

Méthode pour l'utilisation du système promoteur de l'ADH-II de levure pour la production biotechnologique de protéins hétérologues Download PDF

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Publication number
EP0776975B1
EP0776975B1 EP96118309A EP96118309A EP0776975B1 EP 0776975 B1 EP0776975 B1 EP 0776975B1 EP 96118309 A EP96118309 A EP 96118309A EP 96118309 A EP96118309 A EP 96118309A EP 0776975 B1 EP0776975 B1 EP 0776975B1
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EP
European Patent Office
Prior art keywords
hirudin
glucose
culture
yeast
fermentation
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Expired - Lifetime
Application number
EP96118309A
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German (de)
English (en)
Other versions
EP0776975A3 (fr
EP0776975A2 (fr
Inventor
Werner Dr. Badziong
Paul Dr. Habermann
Jörg Dr. Möller
Werner Dr. Aretz
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Bayer Pharma AG
Original Assignee
Schering AG
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Publication date
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Publication of EP0776975A2 publication Critical patent/EP0776975A2/fr
Publication of EP0776975A3 publication Critical patent/EP0776975A3/fr
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Publication of EP0776975B1 publication Critical patent/EP0776975B1/fr
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Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P21/00—Preparation of peptides or proteins
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/80—Vectors or expression systems specially adapted for eukaryotic hosts for fungi
    • C12N15/81—Vectors or expression systems specially adapted for eukaryotic hosts for fungi for yeasts
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/575—Hormones
    • C07K14/5759—Products of obesity genes, e.g. leptin, obese (OB), tub, fat
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/575—Hormones
    • C07K14/62—Insulins
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/81—Protease inhibitors
    • C07K14/815—Protease inhibitors from leeches, e.g. hirudin, eglin
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P21/00—Preparation of peptides or proteins
    • C12P21/02—Preparation of peptides or proteins having a known sequence of two or more amino acids, e.g. glutathione
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide

Definitions

  • the invention relates to a new method for using the yeast p ⁇ -ADH II promoter system for the biotechnological production of hirudin, hirudin derivatives or Fusion proteins containing hirudin or a hirudin derivative.
  • hirudin are peptide-like thrombin inhibitors with a specific Activity of at least 10,000 AT-U / mg (anti-thrombin units / mg) understand that of the well-known isohirudins of the species Hirudo medicinalis are derived and essential structural features of this, in particular the characteristic linkage of the three disulfide bridges (J. Dodt et al., Biol. Chem. Hoppe-Seyler 366 (1985) 379-385), (see e.g.
  • the gene for alcohol dehydrogenase isoenzyme II (ADH II) is in yeast cells strictly regulated.
  • the ADH II gene product is not discovered if the Fermentation medium fermentable carbon sources such as e.g. glucose are clogged.
  • Induction conditions for the ADH II promoter can also be in simple aerobic batch processes can be achieved in shake flasks or in fermenters, e.g. be started with a glucose concentration of 4%.
  • First is due to the so-called crabtree effect when growing on glucose With the help of the enzyme ADH I ethanol is formed, which in turn after complete Consumption of glucose serves as another source of carbon. After breaking down the glucose the ethanol-degrading enzyme ADH II is induced and the expression of Hirudin begins.
  • Such batch fermentations usually do not bring those for industrial ones Applications targeted high space / time yield. That is desirable Separation of the fermentation into a growth and a production phase, such as in classic antibiotic fermentations, for example, through a limited fed-batch procedure is achieved in which after establishing a certain cell density C source is replenished to limit growth under optimal physiological conditions with high yield the product (e.g. the Secondary metabolites penicillin) (Hersbach et al .: The Penicillins: Properties, Biosynthesis and Fermentation, in: Biotechnology of Industrial Antibiotics, pp. 45-140, ed. E.J. Vandamme, Marcel Dekker, New York, 1984).
  • the product e.g. the Secondary metabolites penicillin
  • T ⁇ ttrup et al. (Biotechnol. Bioeng., 35, 339-348, 1990) describe one changed fermentation management for the intracellular production of a Human insulin fusion protein that has a glucose feed with it subsequent ethanol feed for optimal induction of the system includes. They almost succeeded compared to a batch process Doubling the volume-related product yield through continuous Glucose feed at a constant rate throughout Fermentation period. A further doubling was eventually achieved achieved that from a certain point in time the glucose feed through a similar one was replaced by ethanol.
  • step 2 by adding a pulse of glucose with subsequent strong ethanol formation to induce expression more strongly due to the crabtree effect, did not bring increased product yield.
  • glucose is for the low cell population in the Excess exists, i.e. when growth is repressed as a result of Crabtree effect ethanol formed. From a certain point in time it is Cell density so high that glucose no longer acts as the sole carbon source is sufficient and mixed growth on glucose and ethanol occurs. Only when then the primary ethanol formed is also used up and more (Depressed) growth is limited by glucose, the ADH II promoter switched on and product formation begins. Compared to the batch process can so Product yield can be tripled.
  • the invention thus relates to a method for biotechnological Fermentation of heterologous proteins in yeast using a main culture with a pre-culture of a yeast strain expressing the heterologous protein can, inoculated, a small amount of glucose immediately after inoculation, e.g. 0.7-1.4, preferably 0.9-1.3 and very particularly preferably 1.1 g Glucose / l culture volume / h continuously fed at a constant rate while of the entire fermentation ensures aerobic culture conditions by the oxygen partial pressure does not drop below 20% of the saturation, the Fermentation ended after 2 days and finally the heterologous protein isolated from the fermentation approach.
  • a small amount of glucose immediately after inoculation e.g. 0.7-1.4, preferably 0.9-1.3 and very particularly preferably 1.1 g Glucose / l culture volume / h continuously fed at a constant rate while of the entire fermentation ensures aerobic culture conditions by the oxygen partial pressure does not drop below 20% of the saturation, the Fermentation ended after 2 days and finally
  • Another object of the invention is a method according to the above Paragraph described, in which the media of the pre and main culture except a 1% glucose addition in the preculture medium, which is preferably in the Inoculating the main culture is practically completely consumed, equivalent and one or more complex components, e.g. Cornsteep, soy flour or Yeast extract included.
  • "Practically completely consumed” here means a glucose concentration ⁇ 100 mg / l.
  • yeast strains used in the methods mentioned can Saccharomyces cerevisiae, preferably Y79 (see EP 0 324 712) or also Kluyveromyces lactis strains.
  • the heterologous protein which can be produced according to the method can be a hirudin, a hirudin derivative, or a fusion protein containing hirudin or a hirudin derivative.
  • derivatives means among other things functional, i.e. biologically active fragments of Starting proteins or mutants with new, particularly advantageous biological Characteristics.
  • Example 1 Production of the expression vector for recombinant production of hirudin in yeast
  • the vector p ⁇ ADH 2 (see Figure 2A of the Price et al. Article) contains adjacent to the restriction enzyme interface Spe 1 towards 3 end of the c-DNA a unique recognition site for the plasmid for the enzyme Nco1.
  • Example 2 Comparison of laboratory approaches for recombinant hirudin production in yeast when using different Carbon sources
  • the main culture is inoculated with 2% inoculum and incubated for 48 hours.
  • Example 3 Comparison of recombinant hirudin production in yeast by Fermentation with or without glucose addition
  • a fermenter with a total volume of 3,600 l, 1,600 l of culture medium, consisting of one or more complex components, e.g. Cornsteep, soy flour, yeast extract etc., without the addition of glucose or others C sources, such as sucrose, sterilized at 121 ° C for 20 minutes.
  • complex components e.g. Cornsteep, soy flour, yeast extract etc.
  • glucose or others C sources such as sucrose
  • the medium of the pre-culture is equivalent to that of the main culture except that 1% glucose is added.
  • Aerobic conditions must be ensured during fermentation; an oxygen partial pressure of pO2 ⁇ 20% must be maintained.
  • the fermentation is complete after 48 ⁇ 2 hours and is stopped by adding 0.225 ⁇ 0.025% benzalkonium chloride, e.g. 0.45 ⁇ 0.05% ®Dodigen 226 (a 50% solution of a mixture of alkyl-dimethyl-benzylammonium chlorides in water).
  • 0.225 ⁇ 0.025% benzalkonium chloride e.g. 0.45 ⁇ 0.05%
  • ®Dodigen 226 a 50% solution of a mixture of alkyl-dimethyl-benzylammonium chlorides in water.
  • the final volume (medium + condensate + preculture + added glucose solution - water loss due to fumigation) is 2,300 ⁇ 50 l.

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  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Genetics & Genomics (AREA)
  • Zoology (AREA)
  • Engineering & Computer Science (AREA)
  • Molecular Biology (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Wood Science & Technology (AREA)
  • Biophysics (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Engineering & Computer Science (AREA)
  • Biotechnology (AREA)
  • Medicinal Chemistry (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Microbiology (AREA)
  • Biomedical Technology (AREA)
  • Mycology (AREA)
  • Toxicology (AREA)
  • Endocrinology (AREA)
  • General Chemical & Material Sciences (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Diabetes (AREA)
  • Plant Pathology (AREA)
  • Physics & Mathematics (AREA)
  • Child & Adolescent Psychology (AREA)
  • Obesity (AREA)
  • Tropical Medicine & Parasitology (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Peptides Or Proteins (AREA)

Claims (8)

  1. Procédé de fermentation biotechnologique de l'hirudine, de dérivés de l'hirudine ou de protéines de fusion renfermant de l'hirudine ou d'un dérivé de l'hirudine dans de la levure en utilisant un système promoteur pα-ADH II, caractérisé en ce que
    a) une culture principale est inoculée avec une préculture d'une souche de levure qui peut exprimer l'hirudine, un dérivé de l'hirudine ou une protéine de fusion renfermant de l'hirudine ou un dérivé de l'hirudine ;
    b) de 0,7 à 1,4 g de glucose/l de volume de culture/h est introduit en continu à une vitesse constante, immédiatement après l'inoculation ;
    c) des conditions de culture aérobies sont assurées ;
    d) la fermentation est terminée après environ 2 jours, et enfin
    e) l'hirudine, le dérivé de l'hirudine ou la protéine de fusion renfermant de l'hirudine ou un dérivé de l'hirudine est isolé du mélange de fermentation.
  2. Procédé selon la revendication 1, dans lequel le glucose est introduit en continu, immédiatement après l'inoculation, à une vitesse constante de 0,9 à 1,3 ou à 1,1 g de glucose/l de volume de culture/h.
  3. Procédé selon la revendication 1, caractérisé en ce que les milieux de la préculture et de la culture principale sont équivalents, mis à part l'addition de 1 % de glucose au stade de la préculture.
  4. Procédé selon l'une quelconque des revendications 1 ou 3, caractérisé en ce que l'addition de 1 % de glucose dans le milieu de préculture est presque entièrement consommée au moment de l'inoculation.
  5. Procédé selon l'une quelconque des revendications 1 à 4, caractérisé en ce que les conditions de culture aérobies sont assurées par une pression partielle d'oxygène d'au moins 20 % de la saturation.
  6. Procédé selon l'une quelconque des revendications 1 à 5, caractérisé en ce que lesdits milieux contiennent un ou plusieurs constituants complexes tels que, par exemple, l'extrait soluble de maïs, la farine de soja ou l'extrait de levure.
  7. Procédé selon l'une quelconque des revendications 1 à 5, caractérisé en ce que ladite souche de levure est une souche de Saccharomyces cerevisiae, de préférence Y79.
  8. Procédé selon l'une quelconque des revendications 1 à 6, caractérisé en ce que ladite souche de levure est une souche de Kluyveromyces lactis.
EP96118309A 1995-11-28 1996-11-15 Méthode pour l'utilisation du système promoteur de l'ADH-II de levure pour la production biotechnologique de protéins hétérologues Expired - Lifetime EP0776975B1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
DE19544233A DE19544233A1 (de) 1995-11-28 1995-11-28 Verfahren zur Nutzung des Hefe-ADH II-Promotorsystems zur biotechnologischen Produktion heterologer Proteine in hohen Ausbeuten
DE19544233 1995-11-28

Publications (3)

Publication Number Publication Date
EP0776975A2 EP0776975A2 (fr) 1997-06-04
EP0776975A3 EP0776975A3 (fr) 1999-09-29
EP0776975B1 true EP0776975B1 (fr) 2004-09-29

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EP96118309A Expired - Lifetime EP0776975B1 (fr) 1995-11-28 1996-11-15 Méthode pour l'utilisation du système promoteur de l'ADH-II de levure pour la production biotechnologique de protéins hétérologues

Country Status (11)

Country Link
US (1) US5866371A (fr)
EP (1) EP0776975B1 (fr)
JP (1) JP3902822B2 (fr)
KR (1) KR100429935B1 (fr)
AT (1) ATE278025T1 (fr)
AU (1) AU711203B2 (fr)
CA (1) CA2191407C (fr)
DE (2) DE19544233A1 (fr)
DK (1) DK0776975T3 (fr)
ES (1) ES2229250T3 (fr)
PT (1) PT776975E (fr)

Families Citing this family (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
IT1294728B1 (it) * 1997-09-12 1999-04-12 Biopolo S C A R L Ceppi di lievito per la riproduzione di acido lattico
US20070031950A1 (en) * 1998-09-11 2007-02-08 Winkler Aaron A Production of D-lactic acid with yeast
CA2374482C (fr) 1999-05-21 2012-09-18 Cargill Dow Llc Methodes et matieres destinees a la synthese de produits organiques
KR100365533B1 (ko) * 2000-05-10 2002-12-18 재단법인 목암생명공학연구소 상처치유 효과를 가진 재조합 거머린의 제조방법 및 이를 함유하는 약학적 조성물
DE10108211A1 (de) * 2001-02-20 2002-08-22 Aventis Pharma Gmbh Verwendung von Fusionsproteinen, deren N-terminaler Anteil aus einem Hirudinderivat besteht, zur Herstellung rekombinanter Proteine über Sekretion durch Hefen
US7405068B2 (en) * 2003-05-02 2008-07-29 Tate & Lyle Ingredients Americas, Inc. Pyruvate producing yeast strain
CA2623751A1 (fr) * 2005-09-22 2007-04-05 Tate & Lyle Ingredients Americas, Inc. Souches ameliorees pour la production d'acides organiques
KR20130000883A (ko) * 2011-06-24 2013-01-03 삼성전자주식회사 클루이베로마이세스 마르시아누스 내에서의 향상된 단백질 생산
EP3791916A4 (fr) 2018-05-09 2021-12-15 Asahi Intecc Co., Ltd. Tube à usage médical

Family Cites Families (16)

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Publication number Priority date Publication date Assignee Title
IT1157340B (it) 1982-11-29 1987-02-11 F I M A Dispositivo per collegare amovibilamente utensili abrasivi all'albero di levigatrici e simili
CA1341417C (fr) 1984-03-27 2003-01-21 Paul Tolstoshev Vecteurs d'expression de l'hirudine, cellules transformees et procede de preparation de l'hirudine
DE3438296A1 (de) * 1984-04-18 1985-11-07 Hoechst Ag, 6230 Frankfurt Neue polypeptide mit blutgerinnungshemmender wirkung, verfahren zu deren herstellung bzw. gewinnung, deren verwendung und diese enthaltende mittel
ATE64956T1 (de) * 1984-06-14 1991-07-15 Ciba Geigy Ag Verfahren zur herstellung von thrombininhibitoren.
DE3429430A1 (de) 1984-08-10 1986-02-20 Hoechst Ag, 6230 Frankfurt Gentechnologisches verfahren zur herstellung von hirudin und mittel zur durchfuehrung dieses verfahrens
DE3445517C2 (de) 1984-12-13 1993-11-18 Ciba Geigy Für ein Hirudin-ähnliches Protein codierende DNA-Sequenz und Verfahren zur Herstellung eines Hirudin-ähnlichen Proteins
DE3506992A1 (de) 1985-02-27 1986-08-28 Plantorgan Werk Heinrich G.E. Christensen, KG, 2903 Bad Zwischenahn Modifizierte hirudine, verfahren zu deren herstellung und pharmazeutische mittel, die diese wirkstoffe enthalten
EP0324712B1 (fr) 1985-04-11 1993-04-07 Hoechst Aktiengesellschaft Dérivé de l'hirudine
FR2593518B1 (fr) 1985-05-02 1989-09-08 Transgene Sa Vecteurs d'expression et de secretion de l'hirudine par les levures transformees
DE3689525D1 (de) * 1985-07-17 1994-02-24 Hoechst Ag Neue Polypeptide mit blutgerinnungshemmender Wirkung, Verfahren zu deren Herstellung bzw. Gewinnung, deren Verwendung und diese enthaltende Mittel.
AU604925B2 (en) * 1988-02-23 1991-01-03 Schering Aktiengesellschaft A hirudin derivative
CA1324969C (fr) * 1988-05-06 1993-12-07 Jeffrey R. Shuster Expression a haut rendement de proteines dans la levure
DE58906966D1 (de) 1988-06-23 1994-03-24 Hoechst Ag Mini-Proinsulin, seine Herstellung und Verwendung.
NZ233255A (en) * 1989-04-11 1993-02-25 Chiron Corp Plasmodium cs protein analogues lacking one or more of the repeat epitopes, and containing at least one nonrepeat flanking epitope
WO1996007744A1 (fr) * 1994-09-08 1996-03-14 Chiron Corporation Procede de production amelioree des facteurs de croissance proches de l'insuline
US5712249A (en) * 1994-09-08 1998-01-27 Ciba-Geigy Corporation Use of insulin-like growth factors I and II for inhibition of inflammatory response

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Publication number Publication date
ES2229250T3 (es) 2005-04-16
KR100429935B1 (ko) 2004-07-31
CA2191407A1 (fr) 1997-05-29
DE59611099D1 (de) 2004-11-04
JPH09163998A (ja) 1997-06-24
AU711203B2 (en) 1999-10-07
JP3902822B2 (ja) 2007-04-11
US5866371A (en) 1999-02-02
EP0776975A3 (fr) 1999-09-29
DE19544233A1 (de) 1997-06-05
AU7199696A (en) 1997-06-05
DK0776975T3 (da) 2005-01-17
EP0776975A2 (fr) 1997-06-04
PT776975E (pt) 2005-01-31
KR970027317A (ko) 1997-06-24
CA2191407C (fr) 2009-01-06
ATE278025T1 (de) 2004-10-15

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